Actin is an ubiquitous protein which has polymerization characteristics that are important to its function in cell structure and various cell motile processes. In earlier work (the first period of NIH support), we demonstrated that ATP-actin polymerization is markedly affected by the nature of the tightly-bound divalent cation (Mg++ or Ca++). We propose to extend these studies to ADP-actin and then proceed to further basic studies of actin nucleation and polymerization. Comparisons will be made of the kinetics and thermodynamics of the polymerization of Mg-ATP-, Ca-ATP-, Mg-ADP- and Ca-ADP-actins. We also intend to extend the applied hydrostatic pressure study of ATP-actin elongation to the polymerization of Mg- and Ca-ADP-actin, and thereby gain insight into the role of actin-bound ATP hydrolysis during the polymerization of ATP-containing actin. Parallel studies with actin having AMP-PNP as bound nucleotide will provide additional information. A fluorescence spectroscopic investigation of the effect of actin-bound nucleotide and divalent cation on the cytochalasin-induced dimer stabilization reaction will be undertaken. Determination of the number of polymer ends in solution will be accomplished during the second grant period, which will enable us to convert the relative rate constants of polymerization and depolymerization of both ATP-and ADP-actin to absolute rate constants. Finally, the proposed research will seek to characterize secondary salt binding to low affinity (weak) sites on monomeric actin. From these proposed studies, a better understanding of the roles of the actin-bound nucleotide and both tightly- and weakly-bound divalent cations in the polymerization properties of actin will be attained.